![Page 1: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/1.jpg)
The long journey of the African trypanosome
Terry PearsonUniversity of Victoria
Doyle Foundation African Trypanosomosis Meeting, 6-7 June 2006, InternationalLivestock Research Institute, Nairobi
![Page 2: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/2.jpg)
Trypanosomes-still a problem
![Page 3: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/3.jpg)
Geographic distribution of tsetse flies, Geographic distribution of tsetse flies, cattle and human sleeping sickness in sub Saharan Africacattle and human sleeping sickness in sub Saharan Africa
Hersey, 2001
• estimated range occupied by tsetse is 9 million km2 of land
• tsetse-transmitted animal trypanosomiasis, Nagana, results in an annual loss of 5 billion dollars due to cattle mortality and loss of agricultural land
• 60 million people are at risk of contracting African sleeping sickness in endemic areas
Trypanosomes are likely toremain as important pathogens
![Page 4: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/4.jpg)
A Brief History
Human sleeping sickness was described by an Arab writer, al Qualquashaudi in the 14th century
Many African cattle-owning tribes knew of the disease in animals for centuries.
In colonial times, the diseases were described in humans and cattle and were spread into theinterior of the continent along exploration routes.
![Page 5: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/5.jpg)
Discovery of trypanosomes:Researcher: Dr. Gabriel Valentin
Professor, Department of AnatomyUniversity of Bern
1841(Funded by The Swiss National Science Foundation)
(time: 1hour: cost: 4 Swiss Francs)
![Page 6: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/6.jpg)
Researcher: Dr. Mike LehaneProfessor, Liverpool School of Tropical Medicine
2006 Funded by taxpayers
(time 8 days: Cost 1200 pounds)
![Page 7: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/7.jpg)
1894
![Page 8: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/8.jpg)
Drawing by Mary Bruce
![Page 9: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/9.jpg)
Drawing by Declan McKeever
(Bloodstream form trypanosome)
![Page 10: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/10.jpg)
First Wave of Research on Tryps
Discovery of trypanosomesLife cyclePathology
Early drugs
![Page 11: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/11.jpg)
Antigenic Variation
Dick LePage 1968-immunologically different surface coats
George Cross 1975-purified Variant Surface Glycoprotein (VSG)
Recombinant DNA Berg/ Gilbert Monoclonal Antibodies Kohler/Milstein2-D gel electrophoresis O’Farrell/AndersonsTrypanosome culture Hirumi
![Page 12: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/12.jpg)
The Second Wave of Research on Trypanosomes
(Mid 1970s)
Rockefeller Foundation“Great Neglected Diseases Program”
WHO“Special Program for Research and Training in Tropical Diseases”
CGIAR-ILRAD
![Page 13: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/13.jpg)
What was the result?-trypanosome culture-VSG structure-mechanisms: antigenic variation-immune perturbations of the mouse-initiation of the study of bovine immune system-trypanosome differentiation-sensitivity of trypanosomes to serum killing-rudimentary diagnostic tests-identification of a few non-VSG molecules-rudimentary attempts at immunization-training of people
Donor fatigueLoss of faith (research administrators, agencies, some researchers) Decentralization of research into small labs
![Page 14: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/14.jpg)
African trypanosomes in human blood
VSG: an immunological and intellectual barrier
![Page 15: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/15.jpg)
Third (slow)Wave of Trypanosome Research
![Page 16: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/16.jpg)
VSG+/Procyclin - Procyclin+/VSG-
![Page 17: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/17.jpg)
Two classes of Procyclins
![Page 18: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/18.jpg)
The “Spiny Norman” model of procyclic trypanosome surface molecules
![Page 19: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/19.jpg)
Bacteriome
![Page 20: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/20.jpg)
Tsetse-Trypanosome Interactions
![Page 21: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/21.jpg)
Identification of proteins from 2-D gels
Excise spot, wash, reduce, alkylate, digest
Run gel, stain,scan
Extract peptides-mass analyze
Database search
![Page 22: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/22.jpg)
1-D and 2-D gels of midguts from Glossina m. morsitans
N-terminalMicrobial HSP-60:(Wigglesworthia)
N-terminal(microbialOMP)
![Page 23: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/23.jpg)
EP midgut protein:
-increased expression in tsetse midgut after antigen challenge
![Page 24: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/24.jpg)
A strategy for interference with trypanosome transmission:
Express anti-tryps/tsetse molecules in Sodalis (transfection in vitro)
Infect tsetse with engineered Sodalis
Sodalis is transferred to offspring via milk gland secretions
Drive engineered Sodalis into tsetse populations
![Page 25: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/25.jpg)
Anti-trypanosome effectors:
-midgut lectins-midgut EP protein
-anti-procyclin mAbs-antimicrobial peptides
Could these be expressed in tsetse?(by paratransgenesis)
![Page 26: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/26.jpg)
Trypanosoma brucei(bloodstream forms)
Untreated BMAP-18 treated
Trypanosoma brucei(procyclic forms)
Leishmania donovani(promastigotes)
![Page 27: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/27.jpg)
Bloodstream form Bloodstream form Trypanosoma Trypanosoma bruceibrucei treated with treated with antimicrobial peptide BMAPantimicrobial peptide BMAP--1818
![Page 28: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/28.jpg)
Flagellar Pocket
-endocytosis-exocytosis-VSG recycling
VSG recycled every 7 minutesVSG translocation 45 minutes(VSG expressway)
Specific targeting of endocytic pathway-single chain Abs or other targeting ligands? Coupled with ant-trypanosomal peptides?
![Page 29: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/29.jpg)
Fourth Wave:Genome and Post genome
![Page 30: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/30.jpg)
Serengeti 1977
![Page 31: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/31.jpg)
Protein Discovery:
-life cycle stages-serum sens vs serum resistant
-species specific antigens-biomarkers for diagnosis
-biomarkers for tsetse interactions-biomarkers for host responses
iTRAQ
![Page 32: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/32.jpg)
Biomarker Validation:
Stable Isotope Standards and Capture by Anti-Peptide Antibodies
(SISCAPA)
![Page 33: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/33.jpg)
Proteins Measured Clinically in Plasma Span > 10 Orders of Magnitude in Abundance
(199 proteins, literature values)
PPI
![Page 34: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/34.jpg)
SISCAPA*: A New Method Combining The Specificity of MS Detection with Sensitivity of
Antibody Capture
* patent pending
*Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). 2004. Anderson, N.L., et al Journal of Proteome Research 3;235-244.
![Page 35: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/35.jpg)
Multiple-Reaction Monitoring (MRM): Specific Mass Spectrometric Assay for Peptides
• MRM – is a 2-stage MS assay capable of absolute analyte specificity with high precision (CV < 10%). – measures selected peptides in a sample digest as quantitative surrogates for the proteins from which they derive– assays for peptides can be designed directly from protein sequence
MRMs are implemented using triple-quadrupole mass spectrometers (QqQMS), very widely used for small molecular assays in plasma (drug metabolites, inborn errors, pesticides)
Assays can be multiplexed (>100 per run)
![Page 36: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/36.jpg)
LC-MS/MS as Universal Antibody
• SISCAPA is effectively a sandwich immunoassay for a target peptide, in which the second antibody is replaced by a mass spectrometer (acting as a generic second antibody applicable to all analytes and with absolute structural specificity).
Target Target
Sandwich immunoassay
SISCAPA assay
![Page 37: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/37.jpg)
Unfractionated x10047 MRM’s
albuminLVNEVTEFAK
hemopexinNFPSPVDAAFR
α1antichymotrypsinEIGELYLPK
α1antichymotrypsinEIGELYLPK
hemopexinNFPSPVDAAFR
AlbuminLVNEVTEFAK
Unfractionated x1
SISCAPA: Ab3 Hx400-fold enrichment
SISCAPA: Ab4 AAC4,400-fold enrichment
SISCAPA: Specific Enrichment of Peptides from a Plasma Digest
![Page 38: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/38.jpg)
Eye of Science/ Photo Researchers
Thanks!
Isabel RoditiPeter ButikoferSerap AksoyRon GoodingMike LehaneJody HaddowLee HainesAngela Jackson
UVIC ProteomicsLeigh Anderson
Gabrielle & Margaret(Doyle Foundation)
![Page 39: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/39.jpg)
If God had amused himself inventing the lilies of the field, He surely knocked His own socks off with the African parasites
Barbara KingsolverThe Poisonwood Bible
![Page 40: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/40.jpg)
mAb 4A2 (teneral G. m. morsitans midgut)
10nm
![Page 41: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/41.jpg)
mAb 4A2 (teneral G. m. morsitans midgut)
10 nm
![Page 42: The long journey of the African trypanosome 2006 Long Journey.p… · Trypanosoma brucei (bloodstream forms) Untreated BMAP-18 treated Trypanosoma brucei (procyclic forms) Leishmania](https://reader033.vdocuments.net/reader033/viewer/2022053100/605bbe6c204e917e1424cca9/html5/thumbnails/42.jpg)
Could tsetse molecules serve as targets?
Midgut molecules
1. secreted proteins
2. structural proteins